Polymerase Chain Reaction Clonality Assays Based on X-Linked Genes

During embryogenesis in females, in each cell either the paternal or the maternal X chromosome is randomly inactivated through methylation (1 2 ). This event is stably inherited by daughter progeny of each cell. Therefore, in adult women, polyclonal cell populations will comprise a random mixture of paternally and maternally derived X-inactivated cells, but monoclonal cells will contain the same inactivated X chromosome as their progenitor. The paternal and maternal X chromosomes can be distinguished by identification of polymorphisms at certain alleles, and differences in methylation of DNA sequences within these alleles can be detected by digestion of the DNA with methylation-sensitive restriction enzymes. Based on these principles, researchers have developed several X-linked clonality assays, and most are performed using Southern blot hybridization (3 –5 ).